New York University School of Medicine 23 articles published in JoVE Biology Laser Micro-Irradiation to Study DNA Recruitment During S Phase Bearach Miwatani-Minter1,2, Gergely Rona1,2,3 1Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 2Laura and Isaac Perlmutter Cancer Center, New York University School of Medicine, 3Howard Hughes Medical Institute, New York University School of Medicine This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation. Genetics Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator Kayli Sieber1, Maya Saar1, Comzit Opachaloemphan2, Matthew Gallitto3, Huan Yang2, Hua Yan1,4 1Department of Biology, University of Florida, 2Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 3Department of Radiation Oncology, Columbia University Medical Center, 4Center for Smell and Taste, University of Florida Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects. Neuroscience Preparation of the Rat Vocal Fold for Neuromuscular Analyses Charles Lenell1,2, Adrianna C. Shembel1, Aaron M. Johnson1 1NYU Voice Center, Department of Otolaryngology-Head & Neck Surgery, New York University School of Medicine, 2Communicative Sciences and Disorders, New York University This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study. Biochemistry A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction Chen Zhang*1, Min Cui*1, Yazhou Cui1, Aubryanna Hettinghouse1, Chuan-ju Liu1,2 1Department of Orthopaedic Surgery, New York University Medical Center, 2Department of Cell Biology, New York University School of Medicine In this study, we enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used mTOR-rapamycin interaction as an exemplary case. Genetics A Method to Study de novo Formation of Chromatin Domains Ozgur Oksuz1,2,3, Danny Reinberg1,2 1Howard Hughes Medical Institute, New York University School of Medicine, 2Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 3Whitehead Institute for Biomedical Research This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems. Medicine The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation Junjie Liang*1,2, Yuanyuan Guo*1,2, Yuxin Zheng1,2, Shijuan Lang1, Hongxin Chen1,2, Yaoyao You1,2, Bryan O’Young3,4, Haining Ou*1,2, Qiang Lin*1,2 1Department of Rehabilitation, The Fifth Affiliated Hospital of Guangzhou Medical University, 2Experiment Education Model Center of Rehabilitation Medicine, Guangzhou Medical University, 3Department of Physical Medicine and Rehabilitation, Geisinger Health System, 4Department of Rehabilitation Medicine, New York University School of Medicine Here, based on a clinician’s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies. Neuroscience Intracranial Pharmacotherapy and Pain Assays in Rodents Erik Martinez1, Haocheng Zhou1, Jing Wang1,2 1Department of Anesthesiology, Perioperative Care and Pain Medicine, New York University School of Medicine, 2Department of Neuroscience and Physiology, New York University School of Medicine Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain. Neuroscience Bilateral Assessment of the Corticospinal Pathways of the Ankle Muscles Using Navigated Transcranial Magnetic Stimulation Charalambos C. Charalambous1,2, Jing Nong Liang3,4, Steve A. Kautz2,5, Mark S. George5,6, Mark G. Bowden2,5,7 1Department of Neurology, New York University School of Medicine, 2Department of Health Sciences and Research, Medical University of South Carolina, 3Department of Physical Therapy, University of Nevada Las Vegas, 4Department of Health Professions, Medical University of South Carolina, 5Ralph H. Johnson VA Medical Center, 6Department of Psychiatry, Medical University of South Carolina, 7Division of Physical Therapy, Medical University of South Carolina The present protocol describes the simultaneous, bilateral assessment of the corticomotor response of the tibialis anterior and soleus during rest and tonic voluntary activation using a single pulse transcranial magnetic stimulation and neuronavigation system. Immunology and Infection In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model Piul S. Rabbani1, Salma A. Abdou1, Darren L. Sultan1, Jennifer Kwong1, April Duckworth1, Daniel J. Ceradini1 1Hansjörg Wyss Department of Plastic Surgery, New York University School of Medicine We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model. Developmental Biology Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle Wenyue Guan1, Lalanti Venkatasubramanian2, Myungin Baek3, Richard S. Mann2, Jonathan Enriquez1 1Institut de Génomique Fonctionnelle de Lyon, ENS de Lyon, CNRS, 2Departments of Biochemistry and Molecular Biophysics, and Neuroscience, Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University, 3Neuroscience Program, NYU School of Medicine Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps. Immunology and Infection Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes Michael Peled1, Marianne Strazza2, Adam Mor2 1Pulmonary Department, The Chaim Sheba Medical Center, 2Columbia Center for Translational Immunology, Department of Medicine, Columbia University Medical Center Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity. Immunology and Infection Zika Virus Specific Diagnostic Epitope Discovery Maite Sabalza1, Cheryl A. Barber1, William R. Abrams1, Richard Montagna2, Daniel Malamud1,3 1Department of Basic Sciences and Craniofacial Development, New York University College of Dentistry, 2Rheonix, Inc., 3Department of Medicine, New York University Langone School of Medicine In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases. Biochemistry Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells Mo Zhou1, Mark R. Philips1 1Langone Medical Center, New York University Here we present protocols for detergent-free homogenization of cultured mammalian cells based on nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. This method is ideal for monitoring the partitioning of peripheral membrane proteins between soluble and membrane fractions. Neuroscience Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space John Odackal*1, Robert Colbourn*2,3, Namrita Jain Odackal4, Lian Tao5, Charles Nicholson5, Sabina Hrabetova2 1Department of Medicine, University of Virginia, 2Department of Cell Biology, SUNY Downstate Medical Center, 3Neural and Behavioral Science Graduate Program, SUNY Downstate Medical Center, 4Division of Neonatology, University of Virginia, 5Department of Neuroscience and Physiology, New York University School of Medicine This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS. Biochemistry Protein Complex Affinity Capture from Cryomilled Mammalian Cells John LaCava1,2, Hua Jiang1, Michael P. Rout1 1Laboratory of Cellular and Structural Biology, The Rockefeller University, 2Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads. Immunology and Infection Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions Marianne Strazza1, Inbar Azoulay-Alfaguter1, Michael Peled1, Adam Mor1,2 1Department of Medicine, New York University School of Medicine, 2Department of Pathology, New York University School of Medicine This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions. Medicine Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases Stephen T. Armenti1, Jonathan P. Greenberg2, R. Theodore Smith1 1Department of Ophthalmology, NYU School of Medicine, 2Harkness Eye Institute, Columbia University The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy. Neuroscience Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue Nicole Haack1, Simone Durry1, Karl W. Kafitz1, Mitchell Chesler2, Christine R. Rose1 1Institute of Neurobiology, Heinrich Heine University Düsseldorf, 2Departments of Physiology and Neuroscience, New York University School of Medicine We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations. Developmental Biology Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells Ieng Lam Lei1, Lei Bu2, Zhong Wang1 1Cardiac Surgery, University of Michigan, 2Leon H Charney Division of Cardiology, New York University School of Medicine In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines. Medicine MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges Hanzhang Lu1, Peiying Liu1, Uma Yezhuvath1, Yamei Cheng1, Olga Marshall2, Yulin Ge2 1Advanced Imaging Research Center, University of Texas Southwestern Medical Center, 2Center for Biomedical Imaging, Department of Radiology, New York University School of Medicine Non-invasive imaging of the brain vasculature’s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI). Neuroscience Subtype-selective Electroporation of Cortical Interneurons Natalia V. De Marco Garcia1,2, Gord Fishell1 1NYU Neuroscience Institute, New York University School of Medicine, 2Brain and Mind Research Institute, Weill Cornell Medical College This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex. Immunology and Infection Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes Marianne Strazza1, Inbar Azoulay-Alfaguter1, Ariel Pedoeem1, Adam Mor1,2 1Department of Medicine, New York University School of Medicine, 2Departments of Pathology, New York University School of Medicine Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes. Immunology and Infection Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells Melissa V. Fernandez1, Elizabeth A. Miller2, Nina Bhardwaj3 1Department of Pathology, New York University School of Medicine, 2Division of Infectious Diseases, Department of Medicine, Mount Sinai Medical Center, 3Division of Hematology and Oncology, Hess Center for Science and Medicine, Mount Sinai Medical Center Dendritic cells (DCs) secrete IL-1β in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1β can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1β expression.
